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United States Patent | 6,172,070 |
Moschel ,   et al. | January 9, 2001 |
The present invention provides 8-substituted O.sup.6 -benzylguanine, 4(6)-substituted 2-amino-5-nitro-6(4)-benzyloxypyrimidine, and 4(6)-substituted 2-amino-5-nitroso-6(4)-benzyloxypyrimidine derivatives which have been found to be effective AGT inactivators, as well as pharmaceutical compositions comprising such derivatives along with a pharmaceutically acceptable carrier. The present invention further provides a method of enhancing the chemotherapeutic treatment of tumor cells in a mammal with an antineoplastic alkylating agent which causes cytotoxic lesions at the O.sup.6 -position of guanine, by administering to a mammal an effective amount of one of the aforesaid derivatives, 2,4-diamino-6-benzyloxy-s-triazine, 5-substituted 2,4-diamino-6-benzyloxypyrimidines, or 8-aza-O.sup.6 -benzylguanine, and administering to the mammal an effective amount of an antineoplastic alkylating agent which causes cytotoxic lesions at the O.sup.6 -position of guanine.
Inventors: | Moschel; Robert C. (Frederick, MD); Pegg; Anthony E. (Hershey, PA); Dolan; M. Eileen (Oak Park, IL); Chae; Mi-Young (Frederick, MD) |
Assignee: | The United States of America as represented by the Department of Health and (Washington, DC); The Penn State Research Foundation (University Park, PA); Arch Development Corporation (Chicago, IL) |
Appl. No.: | 318238 |
Filed: | May 25, 1999 |
Current U.S. Class: | 514/261.1; 514/922 |
Intern'l Class: | A01N 043/54 |
Field of Search: | 514/258,262,266,922 |
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4801710 | Jan., 1989 | MacCoss et al. | 544/244. |
4965270 | Oct., 1990 | Harnden et al. | 514/262. |
5075445 | Dec., 1991 | Jarvest et al. | 544/276. |
5091430 | Feb., 1992 | Moschel et al. | 514/262. |
5352669 | Oct., 1994 | Moschel et al. | 514/258. |
5358952 | Oct., 1994 | Moschel et al. | 514/266. |
5364904 | Nov., 1994 | Farmer et al. | 514/572. |
5525606 | Jun., 1996 | Moschel et al. | 514/262. |
5691307 | Nov., 1997 | Moschel et al. | 514/12. |
5731304 | Mar., 1998 | Baer et al. | 514/183. |
5753668 | May., 1998 | Moschel et al. | 514/572. |
5916894 | Jun., 1999 | Moschel et al. | 514/262. |
5929046 | Jul., 1999 | McMurry et al. | 514/45. |
5958932 | Jun., 1999 | Moschel et al. | 514/262. |
Foreign Patent Documents | |||
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WO 91/13898 | Sep., 1991 | WO. | |
WO 94/29312 | Dec., 1994 | WO. | |
WO 96/04281 | Feb., 1996 | WO. | |
WO 97/20843 | Jun., 1997 | WO. |
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TABLE 1 AGT-Inactivating Activity of 6-Benzyloxypurine, 6(4)- Benzyloxypyrimidine, and 6-Benzyloxy-s-triazine Derivatives ED.sub.50 (.mu.M).sup.a In HT29 In cell-free HT29 Compound extract cells 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine 0.06 0.02 (3d) 2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e) 0.06 0.02 8-aza-O.sup.6 -benzylguanine (2) 0.07 0.06 O.sup.6 -benzyl-8-bromoguanine (1d) 0.08 0.06 O.sup.6 -benzylguanine 0.2 0.05 O.sup.6 -benzyl-8-methyl-guanine (1b) 0.3 0.1 O.sup.6 -benzyl-8-oxoguanine (1c) 0.3 0.15 2,4,5-triamino-6-benzyloxy-pyrimidine (3c) 0.4 0.3 2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine 0.4 0.06 (5b) 2-amino-4-benzyloxy-5-nitropyrimidine (5a) 0.4 0.05 8-amino-O.sup.6 -benzyl-guanine (1a) 0.7 2 2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) 2 0.8 2,4-diamino-6-benzyloxy-s-triazine (6) 4 1.0 2,4-diamino-6-benzyloxy-pyrimidine (3b) 15 5 O.sup.6 -benzyluric acid (4b) 25 45 4-amino-6-benzyloxy-5-nitropyrimidine (3a) 28 8 O.sup.6 -benzyl-2-fluoro-hypoxanthine (4c) 48 12 O.sup.6 -benzylxanthine (4a) 60 35 N.sup.2 -acetyl-O.sup.6 -benzyl-8-oxo-guanine (4d) 65 11 O.sup.6 -benzyl-N.sup.2 -methyl-guanine (4e) 160 60 O.sup.6 -benzyl-N.sup.2,N.sup.2 -dimethyl-guanine (4f) 200 110 .sup.a The effective dose required to produce 50% inactivation in cell-free extracts upon incubation for 30 min or in cells upon incubation for 4 hr. The values for O.sup.6 -benzylguanine are from Moschel et al., J. Med. Chem., 35, 4486-4491 (1992).
TABLE 2 Killing of HT-29 Colon Cancer Cells by BCNU Combined with AGT Inactivators Colony Inactivator Formation Concentration BCNU per 1000 Inactivator (.mu.M) (.mu.M) cells None None 435 .+-. 63 None 40 442 .+-. 34 O.sup.6 -benzylguanine 10 None 431 .+-. 33 10 40 13 .+-. 6 2.5 40 38 .+-. 15 1 40 277 .+-. 25 8-aza-O.sup.6 -benzylguanine (2) 10 None 537 .+-. 48 10 40 2 .+-. 1 1 40 423 .+-. 42 O.sup.6 -benzyl-8-bromoguanine (1d) 10 None 401 .+-. 22 10 40 1 .+-. 0 1 40 299 .+-. 30 O.sup.6 -benzyl-8-oxoguanine (1c) 10 None 401 .+-. 22 10 40 <1 1 40 221 .+-. 15 O.sup.6 -benzyl-8-methylguanine (1b) 10 None 513 .+-. 76 10 40 <1 1 40 230 .+-. 51 O.sup.6 -benzyl-8-aminoguanine (1a) 10 None 504 .+-. 30 10 40 430 .+-. 41 1 40 475 .+-. 26 2,4,5-triamino-6- 10 None 453 .+-. 59 benzyloxypyrimidine (3c) 10 40 3 .+-. 1 1 40 487 .+-. 32 2,4-diamino-6-benzyloxy-5- 2.5 None 528 .+-. 64 nitrosopyrimidine (3d) 2.5 40 <1 1 40 19 .+-. 4 2,4-diamino-6-benzyloxy-5- 2.5 None 438 .+-. 25 nitropyrimidine (3e) 2.5 40 <1 1 40 45 .+-. 4
TABLE 3 Killing of DU-145 Prostate Cancer Cells by BCNU Combined with AGT Inactivators Colony Inactivator Formation Concentration BCNU per 1000 Inactivator (.mu.M) (.mu.M) cells None None 453 .+-. 81 None 40 394 .+-. 76 O.sup.6 -benzylguanine 10 None 462 .+-. 68 10 40 28 .+-. 5 1 40 299 .+-. 18 8-aza-O.sup.6 -benzylguanine (2) 10 None 452 .+-. 72 10 40 28 .+-. 5 1 40 248 .+-. 21 O.sup.6 -benzyl-8-bromoguanine (1d) 10 None 493 .+-. 90 10 40 16 .+-. 3 1 40 267 .+-. 39 O.sup.6 -benzyl-8-oxoguanine (1c) 10 None 379 .+-. 34 10 40 34 .+-. 3 1 40 329 .+-. 43 O.sup.6 -benzyl-8-methylguanine (1b) 10 None 357 .+-. 43 10 40 50 .+-. 7 1 40 306 .+-. 157 O.sup.6 -benzyl-8-aminoguanine (1a) 10 None 380 .+-. 36 10 40 435 .+-. 70 1 40 295 .+-. 45 2,4,5-triamino-6- 10 None 429 .+-. 101 benzyloxypyrimidine (3c) 10 40 57 .+-. 7 1 40 378 .+-. 60 2,4-diamino-6-benzyloxy-5- 2.5 None 403 .+-. 35 nitrosopyrimidine (3d) 2.5 40 7 .+-. 3 1 40 25 .+-. 4 0.25 40 192 .+-. 17 2,4-diamino-6-benzyloxy-5- 2.5 None 407 .+-. 80 nitropyrimidine (3e) 2.5 40 9 .+-. 2 1 40 59 .+-. 6 0.25 40 129 .+-. 26
TABLE 4 Killing of MCF-71 Breast Cancer Cells by BCNU Combined with AGT Inactivators Colony Inactivator Formation Concentration BCNU per 1000 Inactivator (.mu.M) (.mu.M) cells None None 426 .+-. 78 None 40 364 .+-. 60 O.sup.6 -benzylguanine 10 None 455 .+-. 63 10 40 4 .+-. 2 2.5 40 12 .+-. 6 8-aza-O.sup.6 -benzylguanine (2) 10 None 483 .+-. 27 10 40 2 .+-. 1 O.sup.6 -benzyl-8-bromoguanine (1d) 10 None 380 .+-. 109 10 40 3 .+-. 1 2.5 40 4 .+-. 3 O.sup.6 -benzyl-8-oxoguanine (1c) 10 None 522 .+-. 78 10 40 4 .+-. 2 O.sup.6 -benzyl-8-methylguanine (1b) 10 None 376 .+-. 76 10 40 2 .+-. 1 O.sup.6 -benzyl-8-aminoguanine (1a) 10 None 432 .+-. 36 10 40 95 .+-. 8 2,4,5-triamino-6- 10 None 448 .+-. 55 benzyloxypyrimidine (3c) 10 40 12 .+-. 4 2,4-diamino-6-benzyloxy-5- 2.5 None 447 .+-. 87 nitrosopyrimidine (3d) 2.5 40 2 .+-. 1 2,4-diamino-6-benzyloxy-5- 2.5 None 314 .+-. 49 nitropyrimidine (3e) 2.5 40 2 .+-. 1
TABLE 5 Inhibition of Mutant Alkyltransferase Proteins by O.sup.6 -Benzylguanine or 2,4-Diamino-6-benzyloxy-5- nitrosopyrimidine ED.sub.50 value (.mu.M).sup.a 2,4-diamino-6- benzyloxy-5- Protein O.sup.6 -benzylguanine nitrosopyrimidine Control 0.25 0.05 P140A 5 0.1 P138A/P140A 22 0.3 G156A 60 1 .sup.a The concentration needed to inactivate 50% of the activity in 30 minutes.